Transcriptome Sequencing via Random Ligation and Rolling Circle Amplification
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Solution Overview
Problem
Current methods for determining full-length transcriptome sequences are costly and require extensive sequencing reads, making them inefficient and expensive, especially for analyzing single cells or bulk RNA samples.
Innovation Solution
The method involves transcribing RNA into first-strand cDNA, converting it to double-stranded cDNA, randomly ligating these molecules to form linear templates which are then circularized. This circular template is amplified using rolling circle amplification, fragmented, and finally sequenced to obtain full-length transcriptome sequences.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If traditional whole genome sequencing methods are used to determine full-length transcriptome sequences, then comprehensive sequence data can be obtained, but the cost increases and extensive sequencing reads are required
Solution Approach 1:
The method segments the transcriptome sequencing process by: (1) converting RNA to double-stranded cDNA, (2) randomly ligating cDNA molecules to form linear templates, (3) circularizing linear templates to create circular templates, (4) amplifying circular templates via rolling circle amplification, (5) fragmenting amplified products, and (6) sequencing fragmented products. This segmentation allows full-length transcriptome sequencing with fewer sequencing reads compared to traditional whole genome sequencing.
Solution Approach 2:
The method performs preliminary actions before sequencing by: (1) converting RNA to cDNA, (2) ligating cDNA into circular templates, and (3) amplifying circular templates to generate multiple copies. These preliminary steps create a library of circular templates that can be fragmented and sequenced with fewer reads while still providing sufficient data for full-length transcriptome determination and copy number variant analysis.
2Reliability
If traditional whole genome sequencing methods are used, then sufficient data for copy number variant analysis can be obtained, but the cost and time requirements increase
Solution Approach 1:
The method segments the sequencing workflow to focus resources on circular template fragmentation and sequencing, rather than sequencing entire genomes. By segmenting cDNA into circular templates and then fragmenting these for sequencing, the method obtains sufficient copy number variant data with reduced sequencing depth requirements, lowering both time and cost while maintaining reliability.
Solution Approach 2:
The method changes key parameters of the sequencing approach by using circular templates instead of linear genomic DNA, and by fragmenting circular templates to generate diverse sequences from fewer reads. This parameter change in template structure and processing enables reliable copy number variant analysis with reduced sequencing requirements compared to traditional whole genome sequencing.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
This approach reduces the cost and enhances the efficiency of determining full-length transcriptome sequences by utilizing fewer sequencing reads while providing sufficient data for copy number variant analysis, compared to traditional whole genome sequencing methods.
Implementation Method 1
amplifying the circular template to form a linear amplified molecule comprising a plurality of copies of the circular template
Implementation Method 2
randomly ligating double-stranded cDNA molecules into linear templates
Data Source
AI summary
The present disclosure provides methods and systems for producing full-length sequencing information of transcriptomes from single cells or from the bulk. Random ligation and circularization of barcoded or non-barcoded complementary deoxyribonucleic molecules can be used to provide a circular template for amplification and subsequent sequencing.